Nucleic acid ligands to integrins

ABSTRACT

Methods are described for the isolation of nucleic acid ligands to integrins using the SELEX process. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The nucleic acid ligands of the present invention are useful as therapeutic and diagnostic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/024,997, filed Oct. 17, 2002, now U.S. Pat. No. 7,094,535, which is adivisional of U.S. patent application Ser. No. 09/364,543, filed Jul.29, 1999, now U.S. Pat. No. 6,331,394, both of which are entitled“Nucleic Acid Ligands to Integrins”. U.S. patent application Ser. No.09/364,543 is a continuation in part of U.S. patent application Ser. No.09/606,477, filed Jun. 29, 2000, now U.S. Pat. No. 6,465,189, which is acontinuation of U.S. patent application Ser. No. 08/956,699, filed Oct.23, 1997, now U.S. Pat. No. 6,083,696, which is a continuation of U.S.patent application Ser. No. 08/234,997, filed Apr. 28, 1994, now U.S.Pat. No. 5,683,867, all entitled “Systematic Evolution of Ligands byExponential Enrichment: Blended SELEX.” U.S. Pat. No. 5,683,867 is acontinuation in part of U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No.5,475,096, each of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention is directed towards nucleic acid ligands of integrinsisolated using the SELEX process. SELEX is an acronym for SystematicEvolution of Ligands by EXponential Enrichment. This invention relatesto integrin proteins, and methods and compositions for treating anddiagnosing diseases involving integrins.

BACKGROUND OF THE INVENTION

The integrins are a class of heterodimeric integral membrane proteins,one or more of which are expressed by most cell types (Hynes (1992) Cell69:11-25). Some 16 homologous alpha subunits and 8 homologous betasubunits associate in various combinations to yield an extensive familyof receptors. Each integrin heterodimer has a large extracellular domainthat mediates binding to specific ligands. These ligands may includeplasma proteins, proteins expressed on the surface of adjacent cells, orcomponents of the extracellular matrix. Several of the integrins showaffinity for more than one ligand and many have overlappingspecificities (Hynes (1992) Cell 69:11-25). Both the α and β subunitscontribute to a small intracellular domain that contacts components ofthe actin cytoskeleton, thus forming a physical link between proteinsoutside and inside the cell. Integrins play an important role incellular adhesion and migration, and these properties are controlled bythe cell, in part, by modulation of integrin affinity for its ligands(so-called “inside-out” signaling). Conversely, the presence or absenceof integrin ligation provides specific information about the cellularmicroenvironment, and in many instances integrins serve as a conduit forsignal transduction. Ligand binding by an integrin may promote itsincorporation into focal adhesions, the assembly of cytoskeletal andintracellular signaling molecules into supra-molecular complexes, andthe initiation of a cascade of downstream signaling events includingprotein phosphorylation, calcium release, and an increase inintracellular pH (reviewed by Schwartz et al. (1995) Ann. Rev. Cell Dev.Biol. 11:549-99). Such “outside-in” signaling ties into pathwayscontrolling cell proliferation, migration and apoptosis (Stromblad etal. (1996) J. Clin. Invest. 98:426-33; Eliceiri et al. (1998) J. Cell.Biol. 140:1255-63). Integrins have been shown to play a role in suchdiverse physiological settings as embryonic development, wound healing,angiogenesis, clot formation, leukocyte extravasation, bone resorptionand tumor metastasis.

The β₃-containing integrins are among the best studied of the receptorsuperfamily. The β₃ subunit forms heterodimers with either α_(v)(α_(v)β₃) or α_(IIb) (α_(IIb)β₃), While these integrins show substantialoverlap in ligand specificity, they play very different roles in normalphysiology and in disease.

α_(v)β₃ is expressed by activated endothelial cells, smooth musclecells, osteoclasts, and, at a very low level, by platelets. It is alsoexpressed by a variety of tumor cell types. The integrin binds to anumber of plasma proteins or proteins of the extracellular matrix, manyof which are associated with sites of inflammation or wound healing(Albelda (1991) Am. J. Resp. Cell Mol. Biol. 4:195-203). These includevitronectin, fibronectin, osteopontin, von Willebrand factor,thrombospondin, fibrinogen, and denatured collagen Type I (Hynes (1992)Cell 69:11-25). Each of these proteins share a common sequence motif,arginine-glycine-aspartic acid (RGD), that forms the core of theintegrin binding site.

α_(v)β₃ has been most intensely studied in the context of new bloodvessel formation (angiogenesis) where it mediates the adhesion andmigration of endothelial cells through the extracellular matrix.Angiogenesis in adults is normally associated with the cyclicaldevelopment of the corpus luteum and endometrium and with the formationof granulation tissue during wound repair. In the latter case,microvascular endothelial cells form vascular sprouts that penetrateinto the temporary matrix within a wound. These cells transientlyexpress α_(v)β₃ and inhibition of the ligand binding function of theintegrin temporarily inhibits the formation of granulation tissue (Clarket al. (1996) Am. J. Pathol. 148:1407-21). In cytokine-stimulated orunstimulated angiogenesis on the chick chorioallantoic membrane,blockade of α_(v)β₃ with a heterodimer-specific antibody prevents newvessel formation without affecting the pre-existing vasculature (Brookset al. (1994) Science 264:569-71). Furthermore, the loss of adhesivecontacts by endothelial cells activated for angiogenesis induces aphenotype characteristic of apoptotic cells (Brooks et al. (1994) Cell79:1157-64); that is, ligand binding by α_(v)β₃ appears to transmit asurvival signal to the cell. Thus, adhesion and/or signaling mediated byα_(v)β₃ is essential for the formation of new blood vessels.

Solid tumors are unable to grow to significant size without anindependent blood supply. It is currently hypothesized that theacquisition of an angiogenic phenotype is one of the limiting steps inthe growth of primary tumors and of tumors at secondary sites (Folkman(1995) Nat. Med. 1:27-31). In addition, while the vasculature thatpenetrates a tumor mass provides a source of oxygen and nutrients, italso serves as a conduit for metastatic cells to leave the primary tumorand migrate throughout the body. Thus, inhibition of angiogenesis maylimit both the growth and metastasis of cancerous lesions. Inexperimental settings of tumor-induced angiogenesis, inhibition ofligand-binding by endothelial α_(v)β₃ prevented the formation of newblood vessels (Brooks et al. (1994) Cell 79:1157-64; Brooks et al.(1995) J. Clin. Invest. 96:1815-22), and inhibitors of α_(v)β₃ wereshown to reduce the growth of experimental tumors in vivo (Brooks et al.(1995) J. Clin. Invest. 96:1815-22; Carron et al. (1998) Canc. Res.58:1930-5).

α_(v)β₃ is not only expressed by the microvasculature within tumors, butin some cases, is also found on the surface of tumor cells themselves.In particular, expression of α_(v)β₃ integrin has been detected intissue sections from tumors of melanocytic and astroglial origin(Albelda et al. (1990) Canc. Res. 50:6757-64; Gladson and Cheresh (1991)J. Clin. Invest. 88:1924-32), and the level of integrin expression hasbeen correlated with the stage or metastatic potential of the tumor(Albelda et al. (1990) Canc. Res. 50:6757-64; Gladson et al. (1996) Am.J. Pathol. 148:1423-34; Hieken et al. (1996) J. Surg. Res. 63:169-73).Furthermore, melanoma cells grown in vitro in a three-dimensional matrixof denatured collagen undergo apoptosis upon α_(v)β₃ blockade.

Data such as these have driven an interest in inhibitors of α_(v)β₃ forthe treatment of cancer. At present, two such inhibitors are in or nearclinical trial: Vitaxin is a chimeric Fab fragment derived from theα_(v)β₃-specific monoclonal antibody, LM609 (Wu et al. (1998) Proc. Nat.Acad. Sci. 95:6037-42). A phase I trial in late-stage cancer patientshas been completed and no significant treatment-associated toxicitieswere observed (Gutheil et al. (1998) Am. Soc. Clin. Onc.). EMD121974 isa cyclic pentapeptide inhibitor of α_(v)β₃. A Phase I study of thiscompound in Kaposi's sarcoma, brain tumors and solid tumors is scheduledto begin in 1999.

Angiogenesis (and α_(v)β₃) are implicated in the pathology of severalother diseases, including psoriasis (Creamer et al. (1995) Am. J.Pathol. 147:1661-7), rheumatoid arthritis (Walsh et al. (1998) Am. J.Pathol. 152:691-702; Storgard et al. (1999) J. Clin. Invest. 103:47-54),endometriosis (Healy et al. (1998) Hum. Reprod. Update 4:736-40), andseveral proliferative diseases of the eye (Casaroli Marano et al. (1995)Exp. Eye Res. 60:5-17; Friedlander et al. (1996) Proc. Nat. Acad. Sci.93:9764-9; Hammes et al. (1996) Nat. Med. 2:529-33). Inhibition ofintegrin ligand binding in each of these contexts may providesignificant therapeutic benefit.

Atheromatous plaque and restenosis following angioplasty are pathologiescharacterized by thickening of the intima, the innermost layer of thearterial wall. The proliferation and/or migration of smooth muscle cellsinto the neointima with concomitant deposition of fibrous extracellularproteins contributes to vessel wall thickening and subsequent vesselocclusion. Platelets may also contribute to the development ofrestenotic lesions through adhesion to endothelial cells and the releaseof growth factors and cytokines that stimulate the underlying smoothmuscle cell layer (Le Breton et al. (1996) J. Am. Coll. Cardiol.28:1643-51). α_(v)β₃ integrin is expressed on arterial smooth musclecells (Hoshiga et al. (1995) Circ. Res. 77:1129-35) and mediates theirmigration on vitronectin and osteopontin (Brown et al. (1994)Cardiovasc. Res. 28:1815-20; Jones et al. (1996) Proc. Nat. Acad. Sci.93:2482-7; Liaw et al. (1995) J. Clin. Invest. 95:713-24; Panda et al.(1997) Proc. Nat. Acad. Sci. 94:9308-13), both matrix proteins that areassociated with atheroschlerotic tissues in vivo (Brown et al. (1994)Cardiovasc. Res. 28:1815-20; Giachelli et al. (1995) Ann. N. Y. Acad.Sci. 760:109-26; Panda et al. (1997) Proc. Nat. Acad. Sci. 94:9308-13).In addition, α_(v)β₃ expression on endothelial cells, and to a muchlesser extent on platelets, is responsible for at least part of theadhesive interaction between these cell types (Le Breton et al. (1996)J. Am. Coll. Cardiol. 28:1643-51; Gawaz et al. (1997) Circulation96:1809-18). α_(v)β₃ blockade with RGD-containing peptides or amonoclonal antibody was found to limit neointimal hyperplasia in severalanimal models of restenosis following arterial injury,, (Choi et al.(1994) J. Vasc. Surg. 19:125-34; Srivatsa et al. (1997) Cardiovasc. Res.36:408-28; Slepian et al. (1998) Circulation 97:1818-27; Coleman et al.(1999) Circ. Res. 84:1268-76). Furthermore, treatment of patientsundergoing percutaneous coronary intervention with an anti-β3 antibody(Reopro/abciximab/c7E3), which blocks both the platelet fibrinogenreceptor, α_(IIb)β₃, and α_(v)β₃, provided long term reduction in therates of death or myocardial infarction and in the rate of reocclusionof the artery (Lefkovits et al. (1996) Am. J. Cardiol. 77:1045-51), aneffect that may be mediated through inhibition of α_(v)β₃ ligation. Theobservation that α_(v)β₃ is expressed by microvascular smooth musclecells after experimentally-induced focal cerebral ischemia (Okada et al.(1996) Am. J. Pathol. 149:37-44) suggests that this integrin may alsoplay some role in the development of ischemia/reperfusion injury instroke.

Finally, α_(v)β₃ mediates the attachment of osteoclasts to matrixproteins, particularly osteopontin, on the surface of bone. Osteoclastsare responsible for the resorption of bone in normal physiology as wellas in pathological conditions such as osteoporosis. A monoclonalantibody specific for α_(v)β₃ inhibited the binding and resorption ofbone particles by osteoclasts in vitro (Ross et al. (1993) J. Biol.Chem. 268:9901-7). Furthermore, an RGD-containing protein, echistatin,was shown to block parathyroid-stimulated bone resorption in an animalmodel, as monitored by serum calcium levels (Fisher et al. (1993)Endocrin. 132:1411-3). Inhibitors of α_(v)β₃ integrin are thusconsidered of potential utility in treating debilitating bone loss suchas occurs in osteoporosis.

α_(IIb)β₃ (also referred to as GPIIbIIIa) is the major integrin on thesurface of platelets where it mediates the adhesion of activatedplatelets to the plasma protein fibrinogen (Nachman and Leung (1982) J.Clin. Invest. 69:263-9; Shattil et al. (1985) J. Biol. Chem.260:11107-14). During clot formation, fibrinogen dimers cross-linkplatelets to one another through the integrin receptor. α_(IIb)β₃ alsobinds to several other plasma and cell matrix proteins, including vonWillebrand factor, vitronectin, and fibronectin (Faull and Ginsberg(1996) J. Am. Soc. Nephrol. 7:1091-7).

Clot formation is a tightly regulated process that balances the need forrapid response to vascular injury with the risk of aberrant occlusion ofcritical vessels. The α_(IIb)β₃ heterodimer is constitutively expressedon the surface of resting platelets at approximately 80,000 copies percell (Wagner et al. (1996) Blood 88:907-14); however, the affinity ofthe integrin for fibrinogen is very low on these cells. Activation ofplatelets by ADP, epinephrine, collagen or thrombin leads to a dramaticenhancement in integrin ligand binding activity (Bennett and Vilaire(1979) J. Clin. Invest. 64:1393-401; Marguerie et al. (1979) J. Biol.Chem. 254:5357-63), probably accomplished through a conformationalchange in the receptor (Shattil et al. (1985) J. Biol. Chem.260:11107-14; O'Toole et al. (1990) Cell Reg. 1:883-93; Du et al. (1993)J. Biol. Chem. 268:23087-92). In this prototypic example of “inside-out”control of integrin function, cross-linking of platelets through theα_(IIb)β₃-fibrinogen interaction is confined to local sites of plateletactivation.

Inhibitors of α_(IIb)β₃ ligand binding have been primarily explored inthe context of cardiovascular disease (Chong (1998) Am. J. Health Syst.Pharm. 55:2363-86; Topol et al. (1999) Lancet 353:227-31), but may haveapplication in any of a number of indications where thrombus formationis suspected or is likely. Three α_(IIb)β₃ inhibitors have been approvedfor use in patients experiencing acute coronary syndrome and/or inpatients who are undergoing percutaneous coronary intervention. Reopro(Centocor/Eli Lilly) is a humanized murine monoclonal antibody Fabfragment with specificity for the β₃ chain of α_(IIb)β₃, Integrilin (CORTherapeutics) is a cyclic heptapeptide based on the integrin bindingsite of barbourin, an α_(IIb)β₃ inhibitory protein derived from snakevenom. Aggrastat (Merck & Co.) is a non-peptide small moleculeantagonist of the integrin. Unlike the small molecule inhibitors, Reoprocross-reacts with α_(v)β₃, a fact which may account for the greaterreduction in long-term rates of death and non-fatal myocardialinfarction associated with its use (see above). A significant effort isunderway to identify new inhibitors of the platelet integrin withcharacteristics not found in the cohort of approved drugs. Specifically,compounds with specificity for the active, ligand-binding conformationof α_(IIb)β₃ may reduce the risk of bleeding complications associatedwith the existing anti-clotting therapies. Orally available compoundswould be particularly useful for longer term therapy of patients at riskfor recurrent myocardial infarction or unstable angina.

Given the role of integrins in the various disease states describedabove, it would be desirable to have high specificity inhibitors ofparticular integrins. The present invention provides such agents.

The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by EXponential enrichment, termed the SELEX process, it hasbecome clear that nucleic acids have three dimensional structuraldiversity not unlike proteins. The SELEX process is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules and is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment,” now abandoned, U.S. Pat. No.5,475,096, entitled “Nucleic Acid Ligands” and U.S. Pat. No. 5,270,163(see also WO 91/19813), entitled “Methods for Identifying Nucleic AcidLigands,” each of which is specifically incorporated by reference hereinin its entirety. Each of these applications, collectively referred toherein as the SELEX Patent Applications, describes a fundamentally novelmethod for making a nucleic acid ligand to any desired target molecule.The SELEX process provides a class of products which are referred to asnucleic acid ligands or aptamers, each having a unique sequence, andwhich has the property of binding specifically to a desired targetcompound or molecule. Each SELEX-identified nucleic acid ligand is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets. The SELEX method applied tothe application of high affinity binding involves selection from amixture of candidate oligonucleotides and step-wise iterations ofbinding, partitioning and amplification, using the same generalselection scheme, to achieve virtually any desired criterion of bindingaffinity and selectivity. Starting from a mixture of nucleic acids,preferably comprising a segment of randomized sequence, the SELEX methodincludes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand-enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific high affinity nucleicacid ligands to the target molecule.

It has been recognized by the present inventors that the SELEX methoddemonstrates that nucleic acids as chemical compounds can form a widearray of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby nucleic acids in biological systems.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, bothentitled “Method for Selecting Nucleic Acids on the Basis of Structure,”describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,”, now abandoned, U.S. Pat. No. 5,763,177, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S.patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX,” describe aSELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, which can be non-peptidic, termed Counter-SELEX. U.S.Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands byEXponential Enrichment: Solution SELEX,” describes a SELEX-based methodwhich achieves highly efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolutionof Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No.5,683,867, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Blended SELEX,” respectively. These applications allow thecombination of the broad array of shapes and other properties, and theefficient amplification and replication properties, of oligonucleotideswith the desirable properties of other molecules.

The SELEX method further encompasses combining selected nucleic acidligands with lipophilic compounds or non-immunogenic, high molecularweight compounds in a diagnostic or therapeutic complex as described inU.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes”. Each of the above described patentapplications which describe modifications of the basic SELEX procedureare specifically incorporated by reference herein in their entirety.

It is an object of the present invention to provide methods that can beused to identify nucleic acid ligands that bind with high specificityand affinity to particular integrins.

It is a further object of the present invention to obtain nucleic acidligands to particular integrins that inhibit the ability of thatintegrin to bind its cognate ligand.

It is a further object of the present invention to obtain integrininhibiting pharmaceutical compositions for controlling thrombosis, tumorangiogenesis, tumor cell migration, proliferative ocular diseases,rheumatoid arthritis, psoriasis, osteoporosis, and restenosis.

It is yet a further object of the invention to obtain imaging agents forthe non-invasive detection of deep vein or arterial thrombi.

SUMMARY OF THE INVENTION

Methods are provided for generating nucleic acid ligands to integrins,particularly to the β₃ integrins. The methods use the SELEX process forligand generation. Particular embodiments describe the isolation ofnucleic acid ligand inhibitors of both α_(v)β₃ and α_(IIb)β₃. Thenucleic acid ligand inhibitors are derived from a library of2′-fluoro-pyrimidine RNA sequences and were selected for high affinitybinding to α_(v)β₃. One of the modified nucleic acid ligands is shown toinhibit the binding of either vitronectin or fibrinogen to both of thepurified integrins in vitro. This nucleic acid ligand binds to thesurface of both resting and activated platelets with equivalent affinityand accumulates at the site of a preformed clot in an animal model ofvenous thrombosis.

The nucleic acid ligands provided by the invention are useful astherapeutic agents for a number of diseases including thrombosis andcancer. The nucleic acid ligands of the instant invention are alsouseful as diagnostic agents for thrombosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the binding of affinity-enriched RNA pools toimmobilized α_(v)β₃. 5′-biotinylated RNA pools were incubated at varyingconcentrations in 96-well microtiter plates coated with integrinα_(v)β₃. Bound RNAs were detected via the biotin moiety by a chromogenicassay. Data are expressed in absorbance units at 405 nm as a function ofinput RNA concentration.

FIG. 2 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:60) topurified integrin α_(IIb)β₃. 5′-biotinylated aptamer 17.16 was incubatedat varying concentrations in microtiter wells coated with eitherintegrin α_(v)β₃ or α_(IIb)β₃. Bound RNA was detected via the biotinmoiety using a chromogenic assay. Data are expressed as the per cent ofthe maximum signal to normalize for differences in protein coating.

FIG. 3 illustrates cross-reactivity of aptamer 17.16 (SEQ ID NO:60) topurified integrin α_(v)β₅. 5′-biotinylated aptamer 17.16 or a controlRNA of similar length and base composition were incubated at varyingconcentrations in microtiter wells coated with either α_(v)β₃ orα_(v)β₅. Bound RNAs were detected via the biotin moiety by a chromogenicassay. Data are expressed in absorbance units at 405 nm as a function ofinput RNA concentration.

FIGS. 4A-C illustrate β3 aptamer inhibition of integrin ligand binding.Biotinylated fibrinogen or vitronectin were incubated in microtiterwells coated with either integrin α_(v)β₃ or α_(IIb)β₃ in the presenceor absence of varying concentrations of ligand binding competitors.Competitors included aptamer 17.16 (SEQ ID NO:60), a control RNA ofsimilar length and base composition, a cyclic RGD peptide (cRGD, seeMaterials and Methods), an α_(v)β₃-specific monoclonal antibody (LM609),or unmodified fibrinogen or vitronectin. Bound ligands were detected viabiotin using a chromogenic assay. Data are expressed in absorbance unitsat 405 nm as a function of input competitor concentration. FIG. 4A showscompetition of vitronectin binding to immobilized α_(v)β₃; FIG. 4B showscompetition of fibrinogen binding to immobilized α_(v)β₃; and FIG. 4Cshows competition of fibrinogen binding to immobilized α_(IIb)β₃. Anestimate of the maximum absorbance value was determined for eachligand/integrin pair in the absence of competitor. The baselineabsorbance value was determined by adding 5 mM EDTA to the incubationbuffer. The maximum and minimum values so determined were FIG. 4A,0.914/0.113; FIG. 4B, 1.042/0.122; FIG. 4C, 0.889/0.128.

FIG. 5 illustrates binding of aptamer 17.16 (SEQ ID NO:60) to activatedor resting human platelets. 5′-fluorescein-conjugated aptamer 17.16 or acontrol RNA of similar length and base composition were incubated atvarious concentrations with resting or thrombin-activated humanplatelets (10⁶I/mL). Incubations were at room temperature in bufferedsaline containing divalent cations, 0.1% BSA and 0.01% sodium azide.Mean fluorescence intensity of the sample was determined by flowcytometry both before and after the addition of EDTA to 5 mM finalconcentration. The difference in fluorescence intensity between the twosamples (the EDTA-sensitive signal) is shown as a function of theconcentration of aptamer or control RNA.

FIG. 6 illustrates biodistribution of [^(99m)Tc]-aptamer 17.16 (SEQ IDNO:60) or control RNA in a rabbit venous clot model. A clot derived fromhuman platelet-rich plasma was generated in situ by temporary isolationof the jugular vein of an anesthetized rabbit. After restoration ofcirculation over the clot, [^(99m)Tc]-labeled aptamer or control RNAwere injected into the bloodstream of the rabbit via the ipsilateral earvein. After one hour, the animal was sacrificed and tissues were weighedand counted in a gamma counter. Accumulation of radioactivity in varioustissues is reported as the percentage of the injected dose per gram wetweight of tissue.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The central method utilized herein for identifying nucleic acid ligandsto Integrins is called the SELEX process, an acronym for SystematicEvolution of Ligands by Exponential enrichment and involves (a)contacting the candidate mixture of nucleic acids with integrins, orexpressed domains or peptides corresponding to integrins, (b)partitioning between members of said candidate mixture on the basis ofaffinity to integrins, and c) amplifying the selected molecules to yielda mixture of nucleic acids enriched for nucleic acid sequences with arelatively higher affinity for binding to integrins.

DEFINITIONS

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided:

As used herein, “nucleic acid ligand” is a non-naturally occurringnucleic acid having a desirable action on a target. Nucleic acid ligandsare often referred to as “aptamers”. The term aptamer is usedinterchangeably with nucleic acid ligand throughout this application. Adesirable action includes, but is not limited to, binding of the target,catalytically changing the target, reacting with the target in a waywhich modifies/alters the target or the functional activity of thetarget, covalently attaching to the target as in a suicide inhibitor,facilitating the reaction between the target and another molecule. Inthe preferred embodiment, the action is specific binding affinity for atarget molecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the nucleic acid ligand isnot a nucleic acid having the known physiological function of beingbound by the target molecule. In the present invention, the target is anintegrin, or portions thereof. Nucleic acid ligands include nucleicacids that are identified from a candidate mixture of nucleic acids,said nucleic acid ligand being a ligand of a given target, by the methodcomprising: a) contacting the candidate mixture with the target, whereinnucleic acids having an increased affinity to the target relative to thecandidate mixture may be partitioned from the remainder of the candidatemixture; b) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; and c) amplifying the increasedaffinity nucleic acids to yield a ligand-enriched mixture of nucleicacids.

As used herein, “candidate mixture” is a mixture of nucleic acids ofdiffering sequence from which to select a desired ligand. The source ofa candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

As used herein, “nucleic acid” means either DNA, RNA, single-stranded ordouble-stranded, and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

“SELEX” methodology involves the combination of selection of nucleicacid ligands which interact with a target in a desirable manner, forexample binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain nucleic acidligands to integrins.

The SELEX methodology is described in the SELEX Patent Applications.

“SELEX target” or “target” means any compound or molecule of interestfor which a ligand is desired. A target can be a protein, peptide,carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen,antibody, virus, substrate, metabolite, transition state analog,cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. withoutlimitation. In this application, the SELEX targets are integrins.

As used herein, “solid support” is defined as any surface to whichmolecules may be attached through either covalent or non-covalent bonds.This includes, but is not limited to, membranes, microtiter plates,magnetic beads, charged paper, nylon, Langmuir-Bodgett films,functionalized glass, germanium, silicon, PTFE, polystyrene, galliumarsenide, gold, and silver. Any other material known in the art that iscapable of having functional groups such as amino, carboxyl, thiol orhydroxyl incorporated on its surface, is also contemplated. Thisincludes surfaces with any topology, including, but not limited to,spherical surfaces and grooved surfaces.

Note that throughout this application, various references are cited.Every reference cited herein is specifically incorporated in itsentirety.

A. Preparing Nucleic Acid Ligands to Integrins.

In the preferred embodiment, the nucleic acid ligands of the presentinvention are derived from the SELEX methodology. The SELEX process isdescribed in U.S. patent application Ser. No. 07/536,428, entitled“Systematic Evolution of Ligands by Exponential Enrichment,” nowabandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands,” andU.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Methods forIdentifying Nucleic Acid Ligands.” These applications, each specificallyincorporated herein by reference, are collectively called the SELEXPatent Applications.

The SELEX process provides a class of products which are nucleic acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

4) Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield acandidate mixture containing one or a small number of unique nucleicacids representing those nucleic acids from the original candidatemixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796 bothentitled “Method for Selecting Nucleic Acids on the Basis of Structure,”describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S.patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX,” all describea SELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588,entitled “Systematic Evolution of Ligands by Exponential Enrichment:Solution SELEX,” describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule. U.S. Pat. No. 5,496,938, entitled“Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describes methods forobtaining improved nucleic acid ligands after SELEX has been performed.U.S. Pat. No. 5,705,337, entitled “Systematic Evolution of Ligands byExponential Enrichment: Chemi-SELEX,” describes methods for covalentlylinking a ligand to its target.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,637,459, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro(2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Chimeric SELEX,” and U.S. Pat. No.5,683,867, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Blended SELEX,” respectively. These applications allow thecombination of the broad array of shapes and other properties, and theefficient amplification and replication properties, of oligonucleotideswith the desirable properties of other molecules.

In U.S. Pat. No. 5,496,938 methods are described for obtaining improvednucleic acid ligands after the SELEX process has been performed. Thispatent, entitled Nucleic Acid Ligarids to HIV-RT and HIV-1 Rev, isspecifically incorporated herein by reference.

One potential problem encountered in the diagnostic use of nucleic acidsis that oligonucleotides in their phosphodiester form may be quicklydegraded in body fluids by intracellular and extracellular enzymes suchas endonucleases and exonucleases before the desired effect is manifest.Certain chemical modifications of the nucleic acid ligand can be made toincrease the in vivo stability of the nucleic acid ligand or to enhanceor to mediate the delivery of the nucleic acid ligand. See, e.g., U.S.patent application Ser. No. 08/117,991, filed Sep. 8, 1993, nowabandoned, and U.S. Pat. No. 5,660,985, both entitled “High AffinityNucleic Acid Ligands Containing Modified Nucleotides”, and the U.S.patent application entitled “Transcription-Free SELEX”, U.S. patentapplication Ser. No. 09/356,578, filed Jul. 28, 1999, each of which isspecifically incorporated herein by reference. Modifications of thenucleic acid ligands contemplated in this invention include, but are notlimited to, those which provide other chemical groups that incorporateadditional charge, polarizability, hydrophobicity, hydrogen bonding,electrostatic interaction, and fluxionality to the nucleic acid ligandbases or to the nucleic acid ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,phosphorothioate or alkyl phosphate modifications, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping. In preferred embodiments of the instantinvention, the nucleic acid ligands are RNA molecules that are 2′-fluoro(2′-F) modified on the sugar moiety of pyrimidine residues.

The modifications can be pre- or post-SELEX process modifications.Pre-SELEX process modifications yield nucleic acid ligands with bothspecificity for their SELEX target and improved in vivo stability.Post-SELEX process modifications made to 2′-OH nucleic acid ligands canresult in improved in vivo stability without adversely affecting thebinding capacity of the nucleic acid ligand.

Other modifications are known to one of ordinary skill in the art. Suchmodifications may be made post-SELEX process (modification of previouslyidentified unmodified ligands) or by incorporation into the SELEXprocess.

The nucleic acid ligands of the invention are prepared through the SELEXmethodology that is outlined above and thoroughly enabled in the SELEXapplications incorporated herein by reference in their entirety. TheSELEX process can be performed using purified integrins, or fragmentsthereof as a target. Alternatively, full-length integrins, or discretedomains of integrins, can be produced in a suitable expression system.Alternatively, the SELEX process can be performed using as a target asynthetic peptide that includes sequences found in an integrin.Determination of the precise number of amino acids needed for theoptimal nucleic acid ligand is routine experimentation for skilledartisans.

In some embodiments, the nucleic acid ligands become covalently attachedto their targets upon irradiation of the nucleic acid ligand with lighthaving a selected wavelength. Methods for obtaining such nucleic acidligands are detailed in U.S. patent application Ser. No. 08/123,935,filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,”now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Photoselection of Nucleic AcidLigands and Solution SELEX” and U.S. patent application Ser. No.09/093,293, filed Jun. 8 1998, entitled “Systematic Evolution of Ligandsby Exponential Enrichment: Photoselection of Nucleic Acid Ligands andSolution SELEX,” each of which is specifically incorporated herein byreference in its entirety.

In preferred embodiments, the SELEX process is carried out usingintegrins attached to polystyrene beads. A candidate mixture of singlestranded RNA molecules is then contacted with the beads. In especiallypreferred embodiments, the single stranded RNA molecules have a2′-fluoro modification on C and U residues, rather than a 2′-OH group.After incubation for a predetermined time at a selected temperature, thebeads are washed to remove unbound candidate nucleic acid ligand. Thenucleic acid ligand that binds to the integrin is then released intosolution, then reverse transcribed by reverse transcriptase andamplified using the Polymerase Chain Reaction. The amplified candidatemixture is then used to begin the next round of the SELEX process.Example 2 illustrates-one possible way of performing the SELEX processusing integrins as targets.

In preferred embodiments, the nucleic acid ligands thus obtained areassayed for their ability to inhibit the interaction of the integrinwith its cognate ligand. In one embodiment, this is performed by firstcoating microtiter plates with the appropriate integrin(s). A ligand forthe integrin, such as vitronectin or fibrinogen, is then biotinylatedand contacted with the coated integrin in the presence of the nucleicacid ligand to be assayed. After incubation for a suitable period oftime, the microtiter plate is washed, and the amount of vitronectin orfibrinogen binding to integrin is quantitated by adding astreptavidin-alkaline phosphatase conjugate, followed by a colorimetricsubstrate for alkaline phosphatase, such as p-nitrophenyl phosphate. Thealkaline phosphatase signal in each well of the plate is thus inverselyproportional to the effectiveness of the nucleic acid ligand as aninhibitor of the interaction between the bound integrin and its cognateligand.

In other embodiments, the nucleic acid ligands can be analyzed usingbinding to human platelets as an assay. This can be done, for example,by fluorescently labelling the nucleic acid ligand by any of thenumerous techniques known in the art. The fluorescent nucleic acidligand can then be contacted with platelets, and the amount of nucleicacid ligand can be quantitated using Fluorescence Activated Cell Sorting(FACS).

The distribution of the nucleic acid ligands of the instant inventioncan also be studied in vivo. In some embodiments, nucleic acid ligandsare labelled with a radiolabel used in the art of radioimaging. Forexample, a nucleic acid ligand can be conjugated to the isotope ^(99m)Tcusing one of a number of techniques known in the art. The radiolabelednucleic acid can then be studied in an animal model of venousthrombosis. For example, a human blood clot can be generated in rabbitvein by first isolating the vein in situ by ligation, and then infusingthe vein with human platelet-rich plasma and heparin to induce theformation of a blood clot. Blood flow through the vein is thenre-established, and the radiolabeled nucleic acid ligand is introducedinto the animals blood supply. The distribution of the radiolabelednucleic acid ligand can then be studied in the rabbit's tissues todetermine whether the nucleic acid ligand has accumulated in the clot,rather than in other areas.

The nucleic acid ligands provided by the instant invention have a numberof potential uses as therapeutic and diagnostic agents. In someembodiments, nucleic acid ligands that inhibit the interaction betweenplatelet-expressed integrins and their cognate ligands are administered,along with pharmaceutically accepted excipients, in order to prevent theformation of blood clots in patients susceptible to deep veinthrombosis. In other embodiments, the nucleic acid ligands are used totreat acute thrombosis formation during and following percutaneouscoronary intervention. In still other embodiments, the nucleic acidligands of the invention are used to treat patients with acute coronarysyndromes such as unstable angina or myocardial infarction.

In other embodiments, radiolabeled nucleic acid ligands toplatelet-expressed integrins are administered to individuals who are toundergo major surgery, or have suffered major trauma. Such nucleic acidligands can function as imaging agents for the detection of thrombi, byshowing sites in the body where large aggregations of platelets arepresent. If a thrombosis is detected by radioimaging at a critical sitein the body, then anticoagulant and thrombolytic treatment—includingtreatment with the inhibitory nucleic acid ligands of the instantinvention—can be given locally. The advantage of using such a nucleicacid ligand imaging agent is that the anticoagulant and thrombolytictreatments—which can cause harm if administered prophylactically byallowing internal bleeding to continue without efficient clotting—can begiven only to those individuals who definitely have a dangerousthrombosis. Moreover, these treatments can be specifically injected atthe site where the thrombosis has been detected by the nucleic acidligand, instead of injecting higher concentrations into the bloodstreamin the hope that some active agent will be carried to all potentialsites of thrombosis.

Nucleic acid ligands to α_(v)β₃ integrin can be used to inhibit tumorgrowth and metastasis. They can also be used to treat ocular diseasesincluding, but not limited to, diabetic retinopathy, retinopathy ofprematurity, and macular degeneration. Other diseases for which α_(v)β₃nucleic acid ligands are useful therapeutic agents include, but are notlimited to, endometriosis, psoriasis, rheumatoid arthritis, stroke,osteoporosis, and restenosis.

EXAMPLES

The following examples are given for illustrative purposes only. Theyare not to be taken as limiting the scope of the invention in any way.

Example 1 Isolation of Integrins and Integrin Ligands

α_(v)β₃ integrin was isolated from human placenta and purified byimmunoaffinity chromatography essentially as described by (Smith andCheresh (1988) J. Biol. Chem. 263:18726-31). In brief, human placentaswere diced and the tissue fragments were extracted in a buffercontaining 100 mM octyl-β-D-glucopyranoside detergent (Calbiochem, SanDiego, Calif.). The extract was cleared by centrifugation and applied toan immunoaffinity column α_(v)β₃-specific monoclonal antibody LM609affixed to Affi-Gel 10, (Chemicon International, Inc., Temecula,Calif.)). Protein bound to the column was eluted with a low-pH bufferand fractions were immediately neutralized and analyzed for integrincontent by SDS-polyacrylamide gel electrophoresis. Integrin-containingfractions were pooled and aliquots of the purified material were storedat −80° C. Purified human α_(v)β₃ was also purchased from ChemiconInternational, Inc, as was human α_(v)β₅ integrin. α_(IIb)β₃ andfibrinogen were purchased from Enzyme Research Laboratories, Inc. (SouthBend, Ind.). Vitronectin was purified from outdated human plasmaaccording to the procedure of (Yatohgo et al. (1988) Cell Struct. Func.13:281-92), using heparin affinity chromatography.

Example 2 Generating Nucleic Acid Ligands to Integrins Using the SELEXMethod

A DNA template library of sequence:5′-ttatacgactcactatagggagacaagaataaac (SEQ ID NO:1)gctcaannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnttcgacaggaggctcacaacaggc-3′was prepared by chemical synthesis. The italicized nucleotidescorrespond to a T7 RNA polymerase promoter. There are 40 n residues(a,g,t, or c). A short DNA primer “3N8”:5′-gcctgttgtgagcctcctgtcgaa-3′(SEQ ID NO:2) was annealed to the template and extended using Klenow DNApolymerase (New England Biolabs, Beverly, Mass.). The double-strandedDNA product served as a product for T7 RNA polymerase transcription(enzyme obtained from Enzyco, Inc., Denver, Colo.) to generate a libraryof random-sequence RNAs. 2′-fluoro-CTP and -UTP were used in place ofthe 2′-OH-pyrimidines.

For application of the SELEX process to α_(v)β₃ integrin, the purifiedprotein was diluted 1000-fold from detergent-containing storage bufferinto 50 mM MES (2-[N-morpholino]ethanesulfonic acid), pH 6.1, 150 mMNaCl, 2 mM CaCl₂, to a final concentration of approximately 0.2 μg/mL.3.2 μ polystyrene particles (IDEXX Laboratories, Inc., Westbrook, Me.)were added to the diluted protein and the mixture was rotated overnightat 4° C. The beads were collected by centrifugation and blocked byincubation in 3% BSA in MES buffer (above) for one hour at roomtemperature. Blocked beads were washed several times by resuspension inbinding buffer (50 mM Tris.HCl, pH 7.4 (at 37° C.), 145 mM NaCl, 4 mMKCl, 1 mM MgCl₂, 2 mM CaCl₂, 0.1 mM MnCl₂, 0.01% BSA). For one round ofselection, integrin-coated beads were mixed with RNA and rotated at 37°C. for 4 hours to allow equilibration of the RNA with the immobilizedprotein. The beads were then collected by centrifugation and washed atleast 5 times in binding buffer by rapid resuspension and pelleting,without additional incubation. RNAs that remained bound to the beadswere eluted overnight at 37° C. in binding buffer plus 100 μM cyclic RGDpeptide (“cRGD”) (GPenGRGDSPCA, Life Technologies, Gibco BRL,Gaithersburg, Md). Eluted RNAs were extracted with phenol, thenchloroform:isoamyl alcohol (24:1), and ethanol precipitated. The RNApellet was resuspended and annealed to primer 3N8 for reversetranscription using avian myeloblastosis virus reverse transcriptase(Life Sciences, Inc., St. Petersburg, Fla.). The cDNA pool was amplifiedby the polymerase chain reaction using the 3N8 primer and primer“5N8“:5′-taatacgactcactatagggagacaagaataaacgctcaa-3′ (SEQ ID NO:3) andT. aquaticus DNA polymerase (Perkin Elmer-Cetus, Foster City, Calif.).Transcription of the PCR product generated an RNA pool to initiate a newround of selection. For the first round of selection 1 nmol of RNA(approximately 6×10¹⁴ sequences) was incubated at 2 μM concentrationwith a volume of bead suspension equivalent to 50 pmol of protein(assuming all the integrin had adsorbed to the beads). In subsequentrounds, the concentration of RNA and protein-coated beads were bothreduced to demand higher affinity binding interactions.

The affinity of individual RNAs and RNA pools for α_(v)β₃ was determinedby titration of biotinylated RNA with a small quantity of immobilizedintegrin. Bound RNA was detected through the biotin moiety. BiotinylatedRNA was prepared according to standard transcription protocols, butincluding a 5-fold molar excess of a 5′-biotin-modified GMP over GTP inthe reaction mixture. Methods for synthesizing 5′-biotin-modifiedguanosine nucleotides are described in WO 98/30720 entitled“Bioconjugation of Oligonucleotides,” specifically incorporated hereinby reference in its entirety. The modified nucleotide is incorporated atthe 5′ end of the transcript in proportion to its representation in theguanosine pool. 96-well microtiter plates (Immulon 2, DynatechLaboratories, Inc., Chantilly, Va.) were coated overnight at 4° C. with100 μL purified α_(v)β₃ at a concentration of 0.25 μg/mL in 20 mMTrisHCl, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, 0.1 mM MnCl₂.Coating concentrations were 0.8 μg/mL for α_(v)β₃ and 0.3 μg/mL forα_(v)β₅. Wells were blocked with 200 μL of a solution of 3% BSA in thesame buffer (1 hour at room temperature) then rinsed 3 times with 200 μLbinding buffer (50 mM TrisHCl, pH 7.5, 137 mM NaCl, 2.7 mM KCl, 1 mMMgCl₂, 2 mM CaCl₂, 0.1 mM MnCl₂, 0.1% BSA). Individual RNAs or RNA poolswere denatured briefly at 93° C. in binding buffer without divalentcations or BSA, then serially diluted in the same buffer. 50 μL bindingbuffer containing 2X-concentrations of divalent cations and BSA wereadded to each well, followed by 50 μL RNA dilution. RNAs were allowed toincubate in the integrin-coated wells at 37° C. for 30-60 minutes.Unbound RNAs were removed by 3 rapid washes in binding buffer. To detectbound RNA, 100 μL of a 1:2500 dilution in binding buffer ofstreptavidin-alkaline phosphatase conjugate (Calbiochem) were incubatedin each well for 30 minutes at room temperature, followed by three rapidwashes, as above. 100 μL/well p-nitrophenyl phosphate (Sigma ChemicalCo., St. Louis, Mo.) was added and incubated at room temperature for 30minutes. Color development was monitored by absorbance at 405 nm.Binding data were fit to an equation that describes the fraction of RNAor protein bound as a function of K_(D), and the total concentrations ofRNA and protein in the binding reaction for both monophasic and biphasicbinding behavior (Green et al. (1996) Biochem. 35:14413-24). A controlRNA corresponding to a sequence-scrambled version of aptamer 7.24:5′-gggagacaagaauaaucgcucaacguugaaugcu (SEQ ID NO:4)gcauuauggaguaauugaccgcuacaucccuuucgac aggaggcucacaacaggc-3′was used to monitor non-specific binding of RNA under the conditions ofthe assay.

After seven rounds of the SELEX process, the amount of RNA specificallybound to the integrin-coated beads had increased substantially (data notshown). Although immobilized α_(v)β₃ showed no detectable affinity forrandom sequence RNA, the Round 7 RNA pool bound with an equilibriumdissociation constant (K_(D)) of approximately 4×10⁻⁷ M (FIG. 1). TheRound 7 affinity-enriched pool was cloned and sequences were determinedfor individual molecules in the mixture. Of 92 sequences obtained, 35(38%) were very highly related to one another, in many cases differingat no more than a single base position. These sequences are collectivelyreferred to as “Family 1.” It is likely that many if not most of theseRNAs derived from a single precursor as a result of errors inreplication during the RT and PCR steps. Another 25 sequences (27%)shared a short motif (CCUGCC) that defined a second sequence family(“Family 2”). The remaining 32 sequences (35%) were not obviouslyrelated to sequences in Families 1 or 2 and were thus termed “orphan”sequences. The large percentage of orphan sequences in the round 7 poolsuggested that a great deal of sequence complexity remained in thepopulation. Therefore, the SELEX process was continued in the hope offurther enriching for high affinity sequences whose representation inthe round 7 pool may have been low. Indeed, a substantial improvement inthe affinity of the RNA pool was observed after 8 additional rounds ofaffinity selection (Round 15, FIG. 1). No further improvement was seenafter two more rounds of selection (Round 17, FIG. 1), so clones wereisolated from the Round 15 and Round 17 RNA pools and the sequences ofindividual isolates were compared to those obtained at Round 7.Twenty-seven of 39 sequences derived from the Round 15 pool (69%) weremembers of the highly conserved sequence family, Family 1. Threesequences (8%) could be grouped with Family 2 and 9 sequences (23%) wereorphans. All of the 18 sequences isolated from the Round 17 pool weremembers of sequence Family 1. Thus, in this case, additional rounds ofthe SELEX process served to focus the RNA population on a singlehigh-affinity sequence family that was already predominate at Round 7.

Table 1 shows the sequences of the major family of 2′-F-pyrimidine RNAswith high affinity for α_(v)β₃ (Family 1). Clone names indicate theselected RNA pool from which each sequence was derived (round 7, round15 or round 17) followed by a unique clone number. Note that in severalcases identical sequences were isolated from different RNA pools; inthese cases, both clone names are given. (Clones 17.12A and B wereisolated as end-to-end inserts in a single plasmid.) Numbers inparentheses indicate the frequency with which a particular sequence wasisolated; if no number is given the clone was obtained only once fromthe selected RNA pool. Sequences of the 5′ and 3′ fixed sequence regionscommon to all of the clones are shown at the top in lower case letters.Gaps have been inserted into many of the sequences to highlight thestrong sequence conservation among most of the clones. The length of therandom sequence region is shown for each RNA, as well as an estimate ofthe K_(D) for binding to immobilized α_(v)β₃, where it was determined(ND=not determined). The K_(D) value provided is generally based on oneor the average of two determinations. Family 2 sequences isolated fromthe α_(v)β₃ SELEX are shown in Table 2. The short motif (CCUGCC) held incommon among all the sequences is indicated in boldface letters. InTable 3, sequences with no obvious relationship to Families 1 or 2 areshown. Groups of similar sequences with only two (7.41 and 7.93) orthree (7.11, 7.82 and 7.101) members are also included in Table 3.

The substantial affinity improvement between rounds 7 and 15 must be duein part to the loss of lower affinity species from the population;however, the introduction of and selection for higher affinity sequencevariants of Family 1 may also have contributed to the overall affinityenrichment of the pool. While the affinity of relatively few sequencesfrom the Round 7 pool were measured, their affinities for immobilizedα_(v)β₃ were generally less than that of RNAs derived from Rounds 15 and17 (Tables 1-3).

Example 3 Specificity of the Nucleic Acid Ligands to Integrins

In general, aptamers selected for high-affinity binding to a particulartarget protein show relatively weak binding to other related proteins,except in cases where the degree of homology is very high (for example,see (Green et al. (1996) Biochem. 35:14413-24; Ruckman et al. (1998) J.Biol. Chem. 273:20556-67)). Significant homology exists within thefamilies of integrin alpha and beta sub-units, and both alpha and betasub-units are shared among members of the integrin superfamily. Thus, itwas of interest to assess the relative affinity of the α_(v)β₃ aptamersfor closely related integrins. The affinities were determined using themethods described above. The Family 1 aptamer 17.16 (SEQ ID NO:60) waschosen as a representative of the major sequence family. FIG. 2 showsthat aptamer 17.16 bound with identical affinity to purified, utilizedα_(v)β₃ and to the platelet integrin, α_(IIb)β₃ in a 96-well platebinding assay. Although these two proteins share the β₃ sub-unit incommon, an alignment of the α_(v) and α_(IIb) amino acid sequences showsonly 36% overall sequence identity (Fitzgerald et al. (1987) Biochem.26:8158-65). Short stretches of exact sequence identity, 5 to 9 aminoacids in length, do occur, primarily within four putativecalcium-binding domains of each a sub-unit. Binding of aptamer 17.16 tointegrin α_(v)β₅ was also tested. The β₅ sub-unit shares 56% sequenceidentity with β₃ and is more closely related to β₃ than other members ofthe beta sub-unit family (McLean et al. (1990) J. Biol. Chem.265:17126-31; Suzuki et al. (1990) Proc. Nat. Acad. Sci. 87:5354-8). Noaptamer binding to immobilized integrin α_(v)β₅ was observed (FIG. 3),although an α_(v)-specific antibody detected the presence of α_(v)β₅protein adsorbed to the surface of the well (data not shown). Together,these data strongly suggest that aptamer 17.16, and by extension theother members of sequence Family 1, bind primarily to the 3 sub-unit ofα_(v)β₃ Furthermore, the high-affinity binding of the aptamer to theplatelet integrin, α_(v)β₃ extends its range of potential application toindications involving detection of platelets or inhibition of theirfunction.

Example 4 Aptamer Inhibition of Ligand Binding to Purified Integrins

While the SELEX process identifies RNA sequences with high affinity fora particular target, the procedure used in this example was designed tobias for the recovery of ligand binding site inhibitors by the inclusionof a cRGD peptide competitor in the elution buffer. To test whetheraptamer 17.16 could block the ligand binding site of α_(v)β₃ orα_(IIb)β₃, purified vitronectin and fibrinogen were biotinylated andincubated with one or both of the immobilized integrins in the presenceor absence of varying concentrations of the aptamer or a non-bindingcontrol RNA. This was done as follows: purified integrin ligands,vitronectin and fibrinogen, were biotinylated according to (Smith et al.(1990) J. Biol. Chem. 265:12267-71). Briefly, proteins were dialyzedinto 0.1 M NaHCO₃, 0.1 M NaCl. N-hydroxysuccinimido-LC-biotin (Pierce)was dissolved at 1 mg/mL in DMSO and added to the protein at a ratio of0.1 mg biotin per 1 mg protein. The reaction was allowed to rotate atroom temperature for 2 hours. Biotinylated proteins were dialyzed intophosphate-buffered saline and their concentrations determined byabsorbance at 280 nm. 96-well microtiter plates were coated as describedabove with either α_(v)β₃ or α_(IIb)β₃ and blocked with BSA. A fixedconcentration of biotinylated ligand (fibrinogen: 6 nM final;vitronectin: 10 nM final) was pre-mixed in binding buffer (see“Measurement of Aptamer Binding Affinities,” above) with varyingconcentrations of aptamer, control RNA, cyclic RGD peptide, antibody, orunmodified ligand. The mixtures were incubated in the integrin-coatedwells for 60 minutes at room temperature. After washing, boundbiotinylated ligand was detected by addition of 100 μL/well 1:500dilution streptavidin-alkaline phosphatase conjugate (Calbiochem) (30minutes at room temperature) followed by 100 μL/well p-nitrophenylphosphate, as described above. Absorbance was read at 405 nm. The datawere fit to an equation that describes mutually exclusive binding of twoligands to a single target species (Gill et al. (1991) J. Mol. Biol.220:307-24). The concentration of competitor that inhibited 50% of themaximum signal above background (IC₅₀) was determined from the fittedcurve.

Known ligand binding inhibitors, including an RGD peptide and theα_(v)β₃-specific antibody LM609, were included as positive controls forthe assay. FIG. 4A shows inhibition of biotinylated vitronectin bindingto immobilized α_(v)β₃. Aptamer 17.16 inhibited the binding interactionwith an IC₅₀ of 4.7 nM while the control RNA showed no inhibition. Bycomparison, the IC₅₀ of RGD peptide inhibition was 1.4 nM and that ofLM609 was 2.7 nM. Unmodified vitronectin inhibited the binding of thebiotinylated material with an IC₅₀ of 59 nM. Similar data were obtainedfor aptamer inhibition of fibrinogen binding to α_(v)β₃ (FIG. 4B) andfor fibrinogen binding to α_(IIb)β₃ (FIG. 4C). IC₅₀ values for the datain FIG. 4B were: 17.16, 9.5 nM; control RNA, not measurable; RGDpeptide, 1.0 nM; LM609, 6.3 nM; unmodified fibrinogen, 43 nM. IC₅₀values for FIG. 4C were: 17.16, 6.5 nM; control RNA, not measurable; RGDpeptide, 21 nM; unmodified fibrinogen, 15 nM. Thus, aptamer 17.16 is aneffective competitor of β₃ integrin ligand binding and, on a molarbasis, has an inhibitory potency nearly equivalent to that of a bivalentantibody.

Example 5 Nucleic Acid Ligand Binding to Human Platelets

Aptamer 17.16 (SEQ ID NO:60) was selected for binding to purified humanα_(v)β₃ adsorbed to the surface of a polystyrene bead. In vitro assaysto measure the affinity of the aptamer for purified β₃ integrins werealso done in the context of hydrophobically-adsorbed protein. Thus, animportant test of aptamer function was to determine its capacity to bindto native protein on the surface of cells. Human platelets were chosenfor this purpose because of their ease of isolation and their high levelof expression of integrin α_(IIb)β₃. Because α_(IIb)β₃ undergoes aconformational change upon platelet activation, binding of the aptamerto both resting and thrombin-activated platelets was tested. This wasdone as follow: fluorescein-conjugated RNA was prepared according to(Davis et al. (1998) Nuc. Acids Res. 26:3915-24). Briefly, RNA wastranscribed in the presence of a 5-fold molar excess of the initiatornucleotide guanosine-5′-O-(2-thiodiphosphate) (Calciochem), followed byconjugation of the gel-purified RNA to 5-iodoacetamidofluorescein(Pierce, Rockford, Ill.). Platelet-rich plasma was prepared fromfreshly-drawn citrated human blood by centrifugation at 1000 rpm for 15minutes in a table top centrifuge. For activated platelets, cells wereincubated for 15 minutes at room temperature at 2×10⁷/mL in calcium- andmagnesium-free Dulbecco's PBS with 2.5 U/mL thrombin and 5 mMGly-Pro-Arg-Pro (GPRP) to inhibit platelet aggregation. Cells werediluted 1:10 into binding buffer (20 mM HEPES, pH 7.5, 111 mM NaCl, 5 mMKCl, 1 mM MgCl₂, 1 mM CaCl₂, 0.1% BSA, 0.01% sodium azide). Restingcells were diluted similarly, without exposure to thrombin or GPRP. Theactivation state of resting and thrombin-treated cells was monitored bystaining with fluorophore-conjugated antibodies to CD61 (β₃ integrinsubunit), which binds to all platelets, and to CD62 (P-selectin), amarker of platelet activation. Antibodies were obtained fromBecton-Dickinson Immunocytometry Systems, San Jose, Calif.Fluorescein-conjugated RNAs were diluted in water to 4 μM and denaturedbriefly at 93° C., then diluted to 2 μM with 2×-concentrated bindingbuffer. RNAs were then serially diluted in binding buffer. Each dilutionwas mixed 1:1 with resting or activated platelets and allowed toincubate in the dark at room temperature for 30 minutes. The incubationmixtures were applied directly to a Becton Dickinson FACSCalibur flowcytometer to determine the mean fluorescence intensity of the sample.Under such equilibrium binding conditions, an estimate of the K_(D) foraptamer binding to the cell surface integrin could be obtained.

Non-specific RNA binding to platelets was measured using a control RNAof similar length and base composition to aptamer 17.16. Non-specificbinding became significant at concentrations above approximately 100 nM.Specific binding of the aptamer was distinguished from non-specificbinding by the addition of 5 mM EDTA to the sample: EDTA had no effecton the binding of the control RNA but reduced aptamer binding to thelevel of the control. Specific binding of the aptamer was thus definedas the difference between the fluorescence intensity of the samplebefore the addition of EDTA (specific+non-specific) and the fluorescenceintensity after the addition of EDTA (non-specific only).

FIG. 5 shows representative data for the EDTA-sensitive component ofaptamer binding to both resting and thrombin-activated human platelets.The maximum binding signal is approximately 2-fold higher to activatedplatelets, consistent with the slightly higher level of α_(IIb)β₃ onsuch cells (Wagner et al. (1996) Blood 88:907-14). However, theestimated K_(D) for aptamer binding to platelets was approximately 10 nMfor both cell populations, equivalent to the value determined forbinding in vitro to purified α_(IIb)β₃. Furthermore, aptamer 17.16 bindsto both resting and activated platelets with an affinity equivalent tothat reported for Reopro (abciximab, chimeric 7E3 Fab), an approvedα_(IIb)β₃ antagonist (Mousa et al. (1998) J. Pharm. Exp. Ther.286:1277-84).

Example 6 Nucleic Acid Ligand Biodistribution in Rabbit Venous ClotModel

To explore the application of a β₃-specific aptamer in clot imaging,aptamer 17.16 was labeled at the 5′ end with technitium-99m (^(99m)Tc)and its biodistribution was monitored in a rabbit model of venousthrombosis. In this model, a clot is generated in situ in the isolatedjugular vein of a rabbit from human platelet-rich plasma. Blood flowacross the clot is re-established and the radiolabeled aptamer (or anon-binding control RNA) are introduced into the bloodstream via theipsilateral ear vein. The distribution of the radiolabel into varioustissues is reported as the per cent of the injected dose per gram oftissue.

The experiment was performed as follows: Aptamer 17.16 and a control RNAof similar length and base composition were transcribed using a 5-foldmolar excess of 5′-(O-hexylamino) guanosine monophosphate. Each RNA wasconjugated to Hi₁₅ at 50 mg/mL aptamer in 30% dimethylformamide with 5molar equivalents of Hi₁₅-NHS buffered in 100 mM NaBorate pH 9.3, for 30minutes at room temperature. The conjugation reactions were washed overa 30,000 molecular weight cut-off filter (Microcon 30, Amicon, Inc.,Beverly, Mass.) to remove excess Hi₁₅ cage. The RNAs were then labeledwith ^(99m)Tc in the following manner: to 1 nmol Hi₁₅-aptamer was added200 μl of 100 mM NaPO₄ buffer, pH 8.5, 23 mg/mL NaTartrate, and 50 μL[^(99m)Tc] pertechnetate (5.0 mCi) eluted from a ⁹⁹Mo column (Syncor,Denver) within 12 hours prior to use. The labeling reaction wasinitiated by the addition of 10 μL 5 mg/mL SnCl₂. The reaction mixturewas incubated for 15 minutes at 90° C. Unreacted ^(99m)Tc was removed byspin dialysis through a 30,000 molecular weight cut-off membrane(Centrex, Schleicher & Schuell) with two 300 μL washes. This labelingprotocol results in 30-50% of the added ^(99m)Tc being incorporated witha specific activity of 2-3 mCi/nmol RNA.

For biodistribution studies, rabbits were anesthetized with isofluorane.A two centimeter section of the right jugular vein was isolated in situand all the branches were ligated. A catheter was inserted into thefacial vein. The isolated vein segment was temporarily ligated above andbelow the catheter. The vein segment was flushed with saline. 1000 USPunits of heparin was administered intravenously. 300-400 μL of freshhuman platelet-rich plasma (citrate) activated with calcium and thrombinwas instilled into the isolated vein segment and allowed to clot. After30 minutes the ligatures were removed and blood flow over the thrombuswas re-established (confirmed by the injection of 200 μl of air into theipsilateral ear vein). [^(99m)Tc]-conjugated aptamer or control RNA wasinjected into the ipsilateral ear vein. At 1 hour the rabbit wasexsanguinated and tissues were weighed and counted in a Wallac 1470gamma counter. The aptamer and control RNA were tested at 1 nmol/kg(approximately 0.03 mg/kg).

For aptamer 17.16, radiolabel accumulated in the clot to a significantdegree by one hour after injection, while similar accumulation was notobserved with the control RNA (FIG. 6). Blood clearance of theradiolabel was apparently rapid and mediated primarily by a renalmechanism as judged by moderate accumulation of radioactivity in thekidney for both the aptamer and control RNA. Thus, aptamers specific forα_(IIb)β₃ or for other proteins expressed at high levels on the surfaceof platelets or within the matrix of a clot will serve as useful agentsfor rapid imaging of thrombi. TABLE 1 αvβ3 Family 1 aptamer sequences.Sequence of variable region SEQ Clone name 5′-gggagacaagaauaaacgcucaa[variable region] Sequence K_(D) ID (# of isolates)uucgacaggaggcucacaacaggc-3′ length (nM) NO 7.3 (2) uucuacguuguuuaagggcuuauaugagcgcauuauaccc 40  22 5 7.6; 17.12A uucaacgcuguuuaagggcuuauaugagcgcguuauaccc 40 ND 6 7.12 uucaacgcuguuuaagggcuuauaugagcgcguuacaccc 40 ND 7 7.24 (5) uucaacgcuguucaagggcuuauaugagcgcguuauaccc 40 170 8 7.25 uucaacgauguuuaagggcuuauaugagcgcguuauaccc 40 ND 9 7.34 uucau gaaguccaagggcuuauaugagcgcguuauaccc 39 ND 10 7.36 (3) uucaacgcugucaaagggcuuauaugagcgccguuauaccc 40 ND 11 7.37 (2) uu aacguuguucaaggguuuauaugagugcguuuauaccc 39 ND 12 7.38 (2) uucaacgcuguccaagggcuuauaugagcgcguuauaccc 40  49 13 7.49 uucaacggauguccaagggcuuuaugagcgcguuauaccc 40 ND 14 7.53uucgacgcuguucaagggcuuauaugagcgcauuauaucc 40 ND 15 7.54 (2) uucgacgcuguucaagggcuuauaugagcgcguuauaccc 40 230 16 7.57 (2) uucgacgauguccaagggcuuauaugagcgcauuauaccc 40 ND 17 7.63 uucaacgcuguucaagggcuuauaugagcgcguuacaccc 40 ND 18 7.64 uuc augauguucaagggcuuauaugagcgcauuauaccc 39 ND 19 7.77 (2) uucaacgauguugaggggcuuauaugagcgcauuauaccc 40 770 20 7.80 uucaacgauguccaagggcuuauaugagcgcauuauaccc 40 ND 21 7.86 uucaacgcuguucaagggcuuaugugagcgcguuauaccc 40 ND 22 7.91 uucaacguuguccaagggcuuauaugagcgcauuauaccc 40 ND 23 7.115uucaacgcuguucaagggcuuauaugagcgcauuauaccc 40 ND 24 7.121 uucaacgauguccaagggcuuauaugagcggauua ccc 38 ND 25 7.124 uucaacac ugugaagggcuuauaugagcgcgucauaccc 39 ND 26 7.127uucaacguuguucaagggcuuauaugagcgcgcguauaccc 40 ND 27 15.2 uucaacguugucaaagggcuuauaugagcggauua ccc 38  6 28 15.3 (3); 17.17 uucaacguuguccaagggcuuauaugagcggauua ccc 38  8 29 15.7 uucuacgaugucaaagggcuuauaugagcggauua ccc 38  5 30 15.8 uucgacgcuguugaagggcuuauacgagcggauua ccc 38  5 31 15.10 uucaacgcuguucaagggcuuauaugagcggauua ccc 38  20 32 15.14 uucaacauuguccaagggcuuauaugagcggauua ccc 38  6 33 15.17 uucaacguugucaaagggcuuauacgggcggauua ccc 38  4 34 15.18 (2) uucaacgc ugugaagggcuuauaugagcggauua ccc 37  2 35 15.20 uucaacgcuguccaagggcuuauaugagcgcauuauaccc 40  20 36 15.27 uucgacuauguccaagggcuuauaugagcggauua ccc 38 ND 37 15.28 uucgacgaugucuaagggcuuauaugagcggauua ccc 38 ND 38 15.40; 17.12B uucaacgcuguugaagggcuuauacgagcggauua ccc 38 ND 39 15.41 uucaacguuguccaagggcuuauacgagcggauua ccc 38 ND 40 15.42; 17.14 (2) uucaacgcuguccaagggcuuauacgagcggauua ccc 38 ND 41 15.46; 17.20 uucgacgc ugugaagggcuuauaugagcggauua ccc 37  40 42 15.47 uucaacguugucaaagggcuuauacgagcggauua ccc 38 ND 43 15.48 uucaacgcuguugaagggcuuauaugagcggauua ccc 38 ND 44 15.49 uucuacguugucuaagggcuuauaugagcggauua ccc 38 ND 45 15.50; 17.3 uucgacgc ugugaagggcuuauacgagcggauua ccc 37  30 46 15.52 uucaacgcuguucaagggcuuauacgagcggauua ccc 38 ND 47 15.53 uucaacgcuguccuagggcuuauaugagcgcaggauaccc 40  70 48 15.55 uucuacgcuguuuaagggcuuauaugagcgaauua ccc 38 ND 49 15.57 uucuacguuguccaagggcuuauaugagcggauua ccc 38 ND 50 15.58 uucgacguuguugaagggcuuauaugagcggauua ccc 38 ND 51 17.1 uucaacgcugucaaagggcuuauauaagcggauua ccc 38 380 52 17.2 (2) uucuacgc ugugaagggcuuauaugagcggauua ccc 37  2 53 17.5 uucgacgc ugugaagggcuuauaugagcggau acaccc 38  5 54 17.7 (2) uucuacgc ugugaagggcuuauacgagcggauua ccc 37  6 55 17.8 uucaacguugucuaagggcuuauaugagcggauua ccc 38  18 56 17.10 uucuacguuguugaagggcuuauaugagcggauua ccc 38 ND 57 17.11 uucuacgc ugugaagggcuuauaugagcgaauua ccc 37  4 58 17.13 uucaacgcuguccaagggcuuauaugggcggauua ccc 38  10 59 17.16 uucaacgc ugugaagggcuuauacgagcggauua ccc 37  8 60

TABLE 2 αvβ3 Family 2 aptamer sequences Clone name Sequence of variableregion SEQ (# of 5′-gggagacaagaauaaacgcucaa [variable region] SequenceK_(D) ID isolates uucgacaggaggcucacaacaggc-3′ length (nM) NO: 7.4GUACCGGAUCGCCCUGCCACGGUAUUUGAGACAUUGAAA 39 ND 61 7.5 (3)GGUAGUAAAUGGACUCCUGCCAUCCAAUACUAUCUCUGAG 40 >1000 62 7.13UGUAGUCGCAUGUCGAGCAGCAAUUCCUGCCAUUGUAGG 39 >1000 63 7.14 (2)UGAAGAACUAGACCUGCCCAAGUCCUUCAUCGUGCUUGCU 40 ND 64 7.27 (2)CGAUUAUACUAUCCCUGCCAGUAGUAAUCAGUGCUAUA 38 ND 65 7.29CGGUGAAGACCUCUAUUAACAACAUGACCUGCCUGCGUUG 40 ND 66 7.32CGCAAAUAUGUUCCUGCCAAAUACGGGCGUUGACGCUAGA 40 ND 67 7.43GGACCCUGCCGAGCACAUUUAUUCUGGUAACUGAGCCCCC 40 ND 68 7.51CGCUGAGAGAAAGCCCUGCCCUUUCAGCUCGAGAGUUAUA 40 ND 69 7.58UGAGAUGCAGUUCCUGCCUGCUGCAUUUCUUAGAGUGUGU 40 ND 70 7.83GAUUAACGGUUAUCCUGCCAACCGAUUAUAAGAGCAUGGA 40 ND 71 7.89UGAGAGACUACAAUAGAACUUAUGUAACCUGCCACAUAGG 40 ND 72 7.97UAGGAAGUGUAACCUGCCUCACGGUCCUAUCGAGUAGUUU 40 ND 73 7.100UGAAAACGCAACCUGCCGGCGUCGUCCUGGGUAAUUUA 40 ND 74 7.104AUAGGGGGUACCUGCCGACCCCAGAAAUAAGCGUGAUU 39 ND 75 7.105UCCUGCCAUAGCGUCUUCAUGUCUGACGUUUGAGUUUCCG 40 ND 76 7.107UCCUAGGUUGGUCCUGCCACAGCUCAAAGGUUUAGCUUCA 40 ND 77 7.109ACAUGCAGACAACCCUGCCUUCUGCGUGGUUUAGGAGUA 39 ND 78 7.120AACCUCAGGCGACCUGCCGCUGUCUGAAGUUCGAGCAUAA 40 ND 79 7.122ACUCAAGACCCUGCCACUAUGUGUUACUGAGUAGGAGCGU 40 ND 80 7.125AUUCGAAAUACGGGUUAAAUCCCUGCCUUUAACACGACA 39 ND 81 15.19UGUAGCCGCAUGUCGAGCAGCAAUUCCUGCCAUUGUAGG 39  770 82 15.21CGGUGAAGACCUCUAUUAACAACAUGACCUGCCUGCGUUG 40  200 83 15.34UCCCACCCUGCCUUGUCUGUUUGAUAGAGACACUGUCCUU 40  190 84

TABLE 3 αvβ3 orphan aptamer sequences Sequence of variable region SEQClone name 5′-gggagacaagaauaaacgcucaa [variable region] Sequence K_(D)ID (# of isolates) uucgacaggaggcucacaacaggc-3′ length (nM) NO 7.1gguuugaaagauugccuguagcuccaaaucuuggugagcu 40 ND 85 7.2ucccgccgauagcuuccacgaagaguuaucuguaaaacaa 36 ND 86 7.11ugagcuccugauuccaaaccuauuccguuucuggu 40 ND 87 7.30acuggacaagucaaucucuccggcuugagacuugguuuac 40 ND 88 7.33 (2)cgagcucuugcuuccaaaccuauuccagacguuu cuggg 40 ND 89 7.41 (2)gcgagccuauugucuaagaugcaccaggccuguuaagcau 40 >1000 90 7.42gccuguacggcgauuaugucuuuaccuuaacuguucc 37 ND 91 7.46uaccaauggcacgaauaacugacuaccccccaaaauggaa 40 ND 92 7.47gcggggcuuugcucaaguguuugcaaacgguaaauuccac 40 ND 93 7.61ccuaccgacguccgccgcuggguuaaccuguaaagucacu 40 ND 94 7.66 (2)gugaaccgauaagcgaaaguaguaccccugcuugacuacu 40 >1000 95 7.67ggagcuccuaguuccaaaccuauuccagaaguuuucugggu 41 ND 96 7.75uaguacgcagucauagcggggcagggacuuucuccgugca 40 ND 97 7.76uuauacugguaugccgccgaccagaauuaauccaaugcgu 40 ND 98 7.82ugagcuccugguuccaaaccuauuccagacguuucagggu 40 ND 99 7.85ucuggccugugacuguagucguuucuucgaguugugacgc 40 ND 100 7.92cucaacgauguccaagggcuuauaugagcgcguuacccc 39 ND 101 7.93gcgagccuauugucuaaqaugcgccaagccuguaaagcau 40 ND 102 7.94gacuagccggccugagauccuuguucgccacacaugcugg 40 ND 103 7.96cuucccccgcaaacacauguuuaguacugggagacuuggg 40 ND 104 7.101ugagcuccugauuccgaaccuauuccagacguuucugggu 40 ND 105 7.102cugauccucuugucauuguacaucucgcag 30 ND 106 7.106uacuaagccuaacaaaagagcggauauuggcgcggcacg 39 ND 107 7.108agucuuaguaguaccgccugcuucuaaccuugggcgcuuu 40 ND 108 7.112ugauuucaugacuuaugccgccggcaugacuucaaugacg 40 ND 109 7.114ucaaaggacggaagugccugugcccgacuaaagaguugag 40 ND 110 7.118cuaucgaucguuuuuucauuucccccugaccaucgccug 39 ND 111 7.123uugucccgcgcagaaacgugacaaaauuuaacacgcaccgu 40 ND 112 7.128uucaacguuguucaagggcuuauaugagcgcguuauaccc 40 ND 113 15.4(4)ugauuucaugacuuaugccgccggcaugacuucaaugacg 40  2000 114 15.5gcauucaaaauuugcgagaacgaauagaaguccgagagcc 40  4000 115 15.13 (2)gcgggauuuuccugaucaucccacugauucggggccuuac 40  790 116 15.39ucaaucucggacuagacuaacgaccuugguugacgcuca 39  410 117 15.43cgccguuaucacgacgugcguucugggcgguacucgcgca 40   45 118

1. A method for the treatment of a disease resulting from plateletactivation, the method comprising administering a biologically-effectiveamount of a nucleic acid ligand to a β₃ integrin.
 2. A method fortreating deep vein thrombosis comprising administering abiologically-effective amount of a nucleic acid ligand to a β₃ integrin.3. A pharmaceutical composition for the treatment of deep veinthrombosis comprising a nucleic acid ligand to a β₃ integrin and apharmaceutically acceptable excipient.
 4. A method for the treatment ofa disease in which α_(v)β₃ activation is a contributing pathogenicfactor, the method comprising administering a biologically-effectivedose of a nucleic acid ligand to α_(v)β₃ and a pharmaceuticallyacceptable excipient.
 5. The method of claim 6 wherein said disease isselected from the group consisting of cancer, diabetic retinopathy,retinopathy of prematurity, macular degeneration, endometriosis,psoriasis, rheumatoid arthritis, stroke, osteoporosis, and restenosis.